EP1617274B1 - System for the microscopic observation and/or detection in a light scanning microscope with linear illumination and use - Google Patents

Abstract

The object (Probe) may be a live single-celled animal. It rests on a horizontal reflective or transparent plane (Probentrager). The illumination light channels (Beleuchtungskanal) are placed in diametrically-opposed positions on either side of the objective lens (Lz) and the observation channel (Beobachtungskanal). A parallel light beam (LS1,LS2) passes through each illumination light channel and is reflected inward by two curved mirrors (R1,R2). The two beams are brought to a focus (P) under the center of the objective.

Description

Stelzer et al. Describe a further development of so-called "theta microscopy" ( Lindek, et al .; Journal of Modern Optics, 1999, vol. 46, no. 5, 843-858 ) in which the detection is at an angle of 90 degrees to the illumination, the so-called "SPIM" (selectice plane illumination microscope) (http://www.focusonmicroscopy.org/2004/abstracts/091 Stelzer.pdf).

• J. Swoger et al., "A confocal fiber-coupled single-lens theta microscope", Review of Scientific Instruments, Vol. 69, Nr. 8, 2956-2963 (1998) ;
• S. Lindek et al., "Single-lens theta microscopy: resolution, efficiency and working distance", Journal of Modern Optics, Vol. 46, Nr. 5, 843-858 (1999) ;
• F.-M. Haar et al., "Developments and Applications of Confocal Theta Microscopy", SPIE Conference on Three-Dimensional and Multidimensional Microscopy, Vol. 3605, 48-54 (1999) .

Further developments in theta microscopy can be found in the following publications:

• Swoger et al., "A confocal fiber-coupled single-lens theta microscope", Review of Scientific Instruments, Vol. 69, No. 8, 2956-2963 (1998). ;
• S. Lindek et al., "Single-lens theta microscopy: resolution, efficiency and working distance," Journal of Modern Optics, Vol. 46, No. 5, 843-858 (1999). ;
• F.-M. Haar et al., Developments and Applications of Confocal Theta Microscopy, SPIE Conference on Three Dimensional and Multidimensional Microscopy, Vol. 3605, 48-54 (1999). ,

The invention defined in claim 1 and its advantages are explained in more detail below:

Deviating from the known theta structure, it is advantageous here to provide reflection at the edge outside the actual observation objective, for example via imaging mirrors with a low numerical aperture, which is mechanically part of the objective in order to achieve a very homogeneous Z resolution along a generated line or area.

After a light branching (splitter) for parallel laser illumination from several sides, parallel rays advantageously pass over a main color splitter, which also acts as in FIG DE10257237 may be formed and a scanning optics on the sample.

The sample is focused on one point with a small numerical aperture of at least one side, resulting in a very flat light cone, which is virtually uniformly distributed inside the sample (constant constriction).

The thickness of the line or surface is adjustable by the focal length / numerical aperture.

From above through the lens, a viewing (detection) of the illuminated points along this light cone with a line or area detector takes place. The depth resolution is given by the focal length / numerical aperture of the reflection from the side. In the case of a line scanner, it can additionally be adjusted by means of a confocal slit diaphragm, which is located in front of the line detector.

The beam splitter is advantageously arranged in the objective pupil (of the observation lens) and has at the edge two points or lines, which serve to reflect the quasi-parallel light beams in the direction of the objective. Otherwise it is permeable to sample light.

An inversion (illumination over small transmissive areas) and observation of the reflected sample light is also possible. The line scanner detects one line.

A line is created in the sample and the fluorescence along that line is mapped onto a line detector. Shadows are avoided by lighting from both sides. In principle, one could only illuminate from one side. To create this line is focused by lateral illumination to the point. Therefore, a circular distribution of small cross-section is provided on the mirror in the objective pupil.

The line generated in the object is moved over the object by the scanner present in the pupil (conjugate plane).

The scanner recaptures the line in the direction of detection and maps it to a line detector.

The tail light from the sample passes through the submirror in the direction of the line detector.

It could also be used directly the edge area of a reflective strip under lighting with two points.

It would lose some efficiency through the continuous strip in the SPIM application. Furthermore, the lens would have to be replaced in the line detection against the above.

For example, in the far field, a cylindrical lens or other suitable optics and the mirrors produce a line of illumination along the y-axis to form an observation surface in the xy plane.

For this purpose, the pupil is focused in the Y direction and thus a line illumination is generated.

The lens has reflectors at least in the field of illumination. The dimensions are dimensioned so that a light band can be transmitted in the wide field, this training also with spot beams from the side is usable (picture at different times on different areas of the mirror).

The mirrors (imaging mirrors) focus parallel rays onto the optical axis of the inner objective. The rear focal plane of the mirrors lies in the objective pupil.

An inner lens is used for observation (detection). In the outer area no optics is required. Only when plane mirrors are provided for the beam deflection in the direction of the sample, an optical effect of the outer ring by corresponding optics with a low aperture is required beforehand.

With the choice of the outer focal length, the optical section thickness (along the optical axis of the inner lens) is adjusted (influencing the beam diameter).

With a vario look, it could be set variably.

The mirror optics may be annular, i. for rotationally symmetrical illumination of the sample from all sides. This arrangement is particularly advantageous in a wide-field detection. When using a line scanner, the illumination of the sample is preferably done with a ring segment, i. from a fixed direction. Along the formed axis, which is perpendicular to the optical axis, this can be done by illumination from one or two opposite directions. The two illumination beams preferably form a common focal point in the sample.

The objective can be designed as an immersion objective. In this case, the space from the sample to the first lens surface, including the reflection optics from the side, is always used.

All points along the line or area through the sample are detected in parallel through the line or in the far field without having to increase the intensity. (eg Raman application, in a point scanner, the sample would be charged by the full power at each point to above the destructive limit and thus heated). If the sample is to be read out at the same frame rate, a reduction in the power is also conceivable. By the parallelization of the sample measurement can For this purpose, the integration time can be increased accordingly so that the measured signal is constant after the longer integration time has elapsed.

The energy input for generating the same signal per sample volume is identical to that of a regular LSM point scanner, since the irradiation direction is in the plane of the optical section to be detected.

There are no higher requirements for the light sources - but a full parallelization can be used.

There is no need for increased energy input to achieve the same SNR as in a line scanner and thus lower sample loading.

The possibility arises of the investigation of weak sample interactions such as e.g. Raman effects.

No special sample preparation is necessary

Particularly advantageously, the invention can be adapted to a line scanner, using the scanner which scans the line over the sample and using the elements for insertion of the illumination or suppression of the detection (beam splitter mirror), in particular advantageous areas of a DE10257237 trained beam splitter can be used.

An approach of the objective according to the invention in a suitable pupil is advantageously possible.

Below is a further description with reference to the drawings:

In Fig. 1, a lens assembly is shown, which consists of a central lens unit Lz, which may be a conventional observation objective of a microscope.

In a housing H, light guides LF are provided outside the lens unit Lz, in which parallel illumination beams Ls1, Ls2 in the direction of the sample initially run parallel to the optical axis A of the observation in Lz. The illumination beams Ls1, Ls2 meet reflectors R1, R2 mounted on the housing H, which may be low-aperture imaging mirrors, and focus the illumination beams in the direction perpendicular to the optical axis of the observation at a point Pin of the optical axis of the objective Lz. R1, R2 may also be plane reflecting mirrors, and light aperture imaging elements may then be provided with low aperture imaging elements, whereby R1, R2 serve only for deflection in the direction of the sample and the focus is generated in the sample by the imaging elements.

Due to the small aperture, the waist of the illumination in the area of the sample runs almost parallel and produces in the sample a thin illumination line, which is imaged into the objective pupil P3.

Lens pupil P3, objective Lz and the sample focus P are here in a 2f arrangement, i. each at a distance of the simple focal distance from each other. As a result, the objective can be used for telecentric scanning, for example, of a lighting line in the sample.

In Fig. 2a, which attaches to the objective pupil P3, a beam splitter T is downstream of a light source LQ, which generates two parallel partial beams Ls1, Ls2, which lies over a lying in a conjugate plane of the objective pupil beam splitter, the opposite at its edge circular reflective sections 2b), reflected and transmitted in parallel via a scanner P2 for moving the illumination beams across the sample in one direction, a scanning optics SO and a tube lens for transmitting an intermediate image ZB onto the objective pupil P3.

Advantageously via the pupil P3 the attachment of the objective according to the invention to the beam path of a line scanner, which has a correspondingly designed beam splitter, as in FIG DE10257237 A21 described already has and its transmitting or reflective surfaces can be used.

Via the scanner (in the pupil P2) of the line scanner, the illumination line described here is moved through the sample.

The observation beam path is dashed, the illumination beam path is drawn continuously. The image of the sample in the intermediate image ZB is descanned via tube lens, scanning optics, scanner and transmitted through the effective for the sample radiation surface of the beam splitter MDB (except the circular reflecting points) by means of Pinholeoptik PO on a (here optional) slit SB in front of a line detector ,

FIG. 3a shows the cross section of the objective pupil on the MDB with the illumination channels BK and the effective area for the observation FB.

In Fig.3b, the illuminated line L is shown in the object plane, is focused on the lens and which is detected by means of detection. The thickness of the line is adjusted by varying the effective numerical aperture NA of the lateral optics focused along the direction of irradiation into the sample. If this NA is reduced, the line width increases accordingly. The manipulation of the numerical aperture can also be effected, for example, by means of a variable ring diaphragm, not shown, in the pupil, arranged around the illumination channel. By moving the scanner P perpendicular to the longitudinal direction (X axis), the line on the sample is displaced vertically in the Y direction.

In Fig. 4a, the arrangement for wide field illumination is shown. Here, a divider for illuminating the sample from two directions of irradiation can be used.

FIG. 4b shows the plane of the objective pupil at the beam splitter MDB in the case of far-field illumination.

This advantageously has two line-shaped opposite transmitting regions B1, B2 at the outer edge, which each transmit a line-shaped region of the illumination (dashed lines) in the direction of the outer region of the objective. These regions are imaged with the reflectors toward the low-aperture sample and form a quasi-parallel, flat, light region of small thickness through the sample. The adjustment of the thickness is again effected by an aperture (not shown) in the pupil, which constricts the pupil of the illumination channel along the x-axis at the location of the pupil.

The objective according to the invention is advantageously connected to the beam path of a line scanner via a pupil P3 as in FIG.

The illumination is focused by a cylindrical lens L in the y direction. An illuminator T (for example, birefringent medium) for generating 2 partial beams may optionally be located in the illumination beam path.

4c shows the spanned light surface in the sample plane (focal plane of the objective)

The sample light (dashed) passes through the beam splitter MDB (reflective) in the direction of a surface detector DEF. A Powell Asphere can optionally be used in front of the cylinder optics ZL1 in Fig. 4 to homogenize the illumination along the y-axis.

The invention described represents a significant expansion of the application possibilities of fast confocal laser scanning microscopes. The significance of such a further development can be read off from the standard cell biological literature and the fast cellular and subcellular processes ¹ described therein and the examination methods used with a plurality of dyes ² .
See for example:
¹ B. Alberts et al. (2002): Molecular Biology of the Cell; Garland Science ,
^1.2 G. Karp (2002): Cell and Molecular Biology: Concepts and Experiments; Wiley Text Books ,
^1.2 R. Yuste et al. (2000): Imaging neurons - a laboratory Manual; Cold Spring Harbor Laboratory Press, New York ,
² RP Haugland (2003): Handbook of Fluorescent Probes and Research Products, 10th Edition; Molecular Probes Inc. and Molecular Probes Europe BV ,

The invention has particular importance for the following processes and processes:

Development of organisms

• ■ Abdul-Karim, M.A. et al. beschreiben 2003 in Microvasc. Res., 66:113-125 eine Langzeitanalyse von Blutgefässveränderungen im lebenden Tier, wobei Fluoreszenzbilder in Intervallen über mehrere Tage aufgenommen wurde. Die 3D-Datensätze wurden mit adaptiven Algorithmen ausgewertet, um die Bewegungstrajektorien schematisch darzustellen.
• ■ Soll, D.R. et al. beschreiben 2003 in Scientific World Journ. 3:827-841 eine softwarebasierte Bewegungsanalyse von mikroskopischen Daten von Kernen und Pseudopodien lebender Zellen in allen 3 Raumdimensionen.
• ■ Grossmann, R. et al. beschreiben 2002 in Glia, 37:229-240 eine 3D-Analyse der Bewegungen von Mikrogliazellen der Ratte, wobei die Daten über bis zu 10 Stunden aufgenommen wurden. Gleichzeitig kommen nach traumatischer Schädigung auch schnelle Reaktionen der Glia vor, so dass eine hohe Datenrate und entsprechendes Datenvolumen entsteht.

The invention described is suitable, inter alia, for the investigation of development processes, which are distinguished above all by dynamic processes in tenths of a second up to the hourly range. Example applications at the level of cell aggregates and whole organisms are described, for example:

• ■ Abdul-Karim, MA et al. describe 2003 in Microvasc. Res., 66: 113-125 a long-term analysis of vascular changes in the living animal, taking fluorescence images at intervals over several days. The 3D data sets were evaluated with adaptive algorithms to represent the movement trajectories schematically.
• ■ Soll, DR et al. describe 2003 in Scientific World Journ. 3: 827-841 a software-based motion analysis of microscopic data of nuclei and pseudopods of living cells in all 3 spatial dimensions.
• ■ Grossmann, R. et al. describe 2002 in Glia, 37: 229-240 a 3D analysis of microglial cell movements of the rat, taking the data up to 10 hours. At the same time, rapid reactions of the glia occur after traumatic injury, resulting in a high data rate and corresponding data volume.

• Analyse lebender Zellen in einer 3D Umgebung, deren Nachbarzellen empfindlich auf Laserbeleuchtung reagieren und die von der Beleuchtung der 3D-ROI geschützt werden müssen;
• Analyse lebender Zellen in einer 3D Umgebung mit Markierungen, die gezielt durch Laserbeleuchtung in 3D gebleicht werden sollen, z.B. FRET-Experimente;
• Analyse lebender Zellen in einer 3D Umgebung mit Markierungen, die gezielt durch Laserbeleuchtung gebleicht und gleichzeitig auch ausserhalb der ROI beobachtet werden sollen, z.B. FRAP- und FLIP-Experimente in 3D;
• Gezielte Analyse lebender Zellen in einer 3D Umgebung mit Markierungen und Pharmaka, die manipulationsbedingte Änderungen durch Laserbeleuchtung aufweisen, z.B. Aktivierung von Transmittern in 3D;
• Gezielte Analyse lebender Zellen in einer 3D Umgebung mit Markierungen, die manipulationsbedingte Farbänderungen durch Laserbeleuchtung aufweisen, z.B. paGFP, Kaede;
• Gezielte Analyse lebender Zellen in einer 3D Umgebung mit sehr schwachen Markierungen, die z.B. eine optimale Balance von Konfokalität gegen Detektionsempfindlichkeit erfordern.
• Lebende Zellen in einem 3D-Gewebeverband mit variierenden Mehrfachmarkierungen, z.B. CFP, GFP, YFP, DsRed, HcRed u.ä.
• Lebende Zellen in einem 3D-Gewebeverband mit Markierungen, die funktionsabhängige Farbänderungen aufweisen, z.B. Ca+-Marker
• Lebende Zellen in einem 3D-Gewebeverband mit Markierungen, die entwicklungsbedingte Farbänderungen aufweisen, z.B. transgene Tiere mit GFP
• Lebende Zellen in einem 3D-Gewebeverband mit Markierungen, die manipulationsbedingte Farbänderungen durch Laserbeleuchtung aufweisen, z.B. paGFP, Kaede
• Lebende Zellen in einem 3D-Gewebeverband mit sehr schwachen Markierungen, die eine Einschränkung der Konfokalität zugunsten der Detektionsempfindlichkeit erfordern.
• Letztgenannter Punkt in Kombination mit den Vorangehenden.

This concerns in particular the following emphases:

• Analysis of living cells in a 3D environment whose neighboring cells are sensitive to laser illumination and need to be protected by 3D ROI lighting;
• Analysis of living cells in a 3D environment with markers to be specifically bleached by laser illumination in 3D, eg FRET experiments;
• Analysis of living cells in a 3D environment with markers that are targeted to be bleached by laser illumination and, at the same time, also observed outside the ROI, eg FRAP and FLIP experiments in 3D;
• Targeted analysis of living cells in a 3D environment with markers and pharmaceuticals exhibiting manipulation-induced changes by laser illumination, eg activation of transmitters in 3D;
• Targeted analysis of living cells in a 3D environment with markings exhibiting manipulation-related color changes by laser illumination, eg paGFP, Kaede;
• Targeted analysis of living cells in a 3D environment with very weak markers that require, for example, an optimal balance of confocality versus detection sensitivity.
• Living cells in a 3D tissue association with varying multiple markings, eg CFP, GFP, YFP, DsRed, HcRed and the like.
• Living cells in a 3D tissue association with markers that have function-dependent color changes, eg Ca + markers
• Living cells in a 3D tissue association with markers showing developmental color changes, eg transgenic animals with GFP
• Living cells in a 3D tissue association with markings that have manipulation-related color changes by laser illumination, eg paGFP, Kaede
• Living cells in a 3D tissue association with very weak markers that require restriction of confocality in favor of detection sensitivity.
• Last point in combination with the previous ones.

Claims (7)

1. Laser scanning microscope for detecting at least one sample region, having

   - a light source which defines an illumination beam path, with

   - at least one scanner (P) for moving an illumination line in a scanning manner through the sample, with

   - a detector (DFEF) in a detection beam path, and with

   - a microscope objective which includes viewing optics (Lz) for viewing at least partially transparent samples,

   characterised in that

   - the microscope objective contains in its housing (H) a light guide (LF) for at least one parallel beam bundle (Lsl) of the illumination outside of the viewing optics (Lz),

   - and at least one reflector (R1) is provided on the objective for diverting the beam bundle, in order to illuminate the sample at least from one side at an angle perpendicular to the optical axis (A) of the viewing optics (Lz), and that

   - in the detection beam path a locally resolving detector (DFEF) is provided for detecting the sample image, wherein

   - in the sample the illumination line is generated perpendicular to the optical axis (A) of the viewing optics, in that the parallel beam bundle is focussed into the sample with a lower aperture than that of the microscope objective and

   - the illumination line is moved by means of the scanner across the object, wherein

   - the sample light in the detection direction is imaged via the viewing optics (Lz) and the scanner (P) on to the locally resolving detector (DFEF).
2. Laser scanning microscope as claimed in claim 1, wherein the reflector (R1) is an imaging mirror which focuses the illumination light in the direction of the sample.
3. Laser scanning microscope as claimed in claim 1,
   wherein the reflector (R1) is a planar mirror and in the light guide an imaging element is provided for focussing purposes.
4. Laser scanning microscope as claimed in any one of claims 1-3, wherein illumination occurs from two sides with a common focal point.
5. Laser scanning microscope as claimed in any one of the preceding claims, wherein the illumination light is coupled-in via a beam splitter (MDB), preferably in the objective pupil, which beam splitter for coupling-in purposes comprises on its periphery slightly extended transmitting or reflective regions with respect to the direction of the illumination light on to the sample, but otherwise is formed substantially on the remainder of the surface so as to reflect or transmit the sample light.
6. Method of detecting at least one sample region using a laser scanning microscope as claimed in any one of claims 1-5, for the examination of weak sample interactions, in particular Raman effects.
7. Method of detecting at least one sample region using a laser scanning microscope as claimed in any one of claims 1-5, for the examination of development processes, in particular dynamic processes in the range of tenths of a second to several hours, in particular at the level of cell groups and whole organisms, in particular according to at least one of the following points:

   • Analysis of live cells in a 3D environment, whose neighbouring cells react sensitively to laser illumination and which must be protected from the illumination of the 3D-ROI;

   • Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination in 3D, e.g. FRET-experiments;

   • Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination and at the same time are also to be observed outside the ROI, e.g. FRAP- and FLIP-experiments in 3D;

   • Targeted analysis of live cells in a 3D environment with markings and medicines which comprise manipulation-induced changes by laser illumination, e.g. activation of transmitters in 3D;

   • Targeted analysis of live cells in a 3D environment with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kaede;

   • Targeted analysis of live cells in a 3D environment with very weak markings which require e.g. an optimum balance between confocality and detection sensitivity.

   • Live cells in a 3D tissue formation with varying multiple markings, e.g. CFP, GFP, YFP, DsRed, HcRed and the like.

   • Live cells in a 3D tissue formation with markings which comprise colour changes which are dependent upon function, e.g. Ca+-markers.

   • Live cells in a 3D tissue formation with markings which comprise development-induced colour changes, e.g. transgenic animals with GFP.

   • Live cells in a 3D tissue formation with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kaede.

   • Live cells in a 3D tissue formation with very weak markings which require a restriction in confocality in favour of detection sensitivity.

Images